Organ Crosstalk

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Professor Dr. Michael Wolf

Klinik für Kieferorthopädie

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+49 241 80-88271

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Regulatory networks controlling the soft tissue-alveolar bone crosstalk in periodontal disease: From a comprehensive mechanistic understanding to novel diagnostic and therapeutic strategies


Univ.-Prof. Dr. Michael Wolf
Department of Orthodontics and Craniofacial Orthopedics

Inflammatory diseases and degenerative processes can cause a loss of alveolar bone mass and periodontal/peri-implant degeneration, resulting in healing defects, tooth destabilization, loss of orthodontic treatment option and ultimately tooth or implant loss.

Often, alterations in oral and periodontal conditions can be a sign of severe systemic disease. However, the molecular mechanisms underlying this sequence of events are currently not well understood, and therefore at present no targeted therapies are available that can efficiently prevent and treat periodontal diseases and tooth destabilization.

This IZKF joint project aims at replacing the conventional tissue centered view on the pathophysiology of periodontal disease by an integrated and interdisciplinary approach that takes into account that dental diseases are influenced not only by complex interactions within oral cavity, but also by systemic changes and diseases. Therefore, scientific groups with a focus on dental diseases and working groups with expertise and strong scientific track records in systemic diseases will join forces to explore mechanisms of organ cross-talk in periodontal disease in this proposal. In the individual projects of this joint proposal, several mechanistic and methodological approaches will be combined.

We aim at generating synergies between projects that will allow a more comprehensive view on mechanisms of organ interactions in periodontal disease by bringing these distinct areas of expertise together. The biological findings and clinical implications of this scientific approach will help to identify novel biomarkers, develop innovative diagnostic tools and therapeutic strategies in this clinical setting.

 

Organ Crosstalk

Here you find the summarys of the subprojects of the Research Area.

OC1-1: The influence of the soft tissue´s Nrf2-Keap1 system on alveolar and long bone regeneration

Univ.-Prof. Dr. rer. nat. Thomas Pufe
Dr. med. Yusuke Kubo
Dr. med. dent. Mohammad Al Asmi     

Inst. für Anatomie und Zellbiologie

 

Reconstituting lost bone is a subject that is germane to many orthopedic conditions including non-union, bone tumors, osteoporosis, periprosthetic infection, or inflammatory arthritis. While acute inflammation appears to be beneficial for bone repair, chronic inflammation is not and dysregulated inflammation increases bone resorption, causing net bone loss. Periodontitis is also characterized by destruction of connective tissue through an inflammatory host response secondary to infection by periodontal bacteria, ultimately causing excessive alveolar bone resorption, which forms a challenge to maxillofacial surgeons.

Crosstalk among inflammatory cells and cells related to bone healing is essential to bone formation, repair and remodeling. Reactive oxygen species (ROS) have a broad role as intra- and intercellular messengers in diverse cell signaling processes and the Keap1-Nrf2 system is a master regulator of the cytoprotective defense.

We were the first to demonstrate a crucial role of Nrf2 in bone regeneration.

We now aim at demonstrating potential differences of Keap1-Nrf2 signaling during the regeneration of two different types of bone (alveolar vs. long bone) as well as elucidating its role in the periodontal ligament.

OC1-2 Dissecting the role of TLR4 dependent signaling networks in vitro and quantitative molecular imaging of periodontal bone remodelling in vivo.

Univ.-Prof. Dr. Michael Wolf 
Department of Orthodontics and Craniofacial Orthopedics

Univ.-Prof. Dr. med. Felix Mottaghy 
Department of Nuclear Medicine

Clinical evidence refers to a fundamental role of periodontal micro-environment for regulating tissue remodeling, tooth movement and progression of tooth root resorption to periodontal breakdown, the most frequent reason for loss of teeth. Recently, toll-like-receptor 4 (TLR4) signaling networks were proposed as potential modulators of benign periodontal remodeling as well as in diseases but the exact roles of TLR-signaling in periodontal tissue for regeneration and degeneration are still unclear.

The project consists of 3 parts:

  • Part (I) addresses the regulation of TLR4 in an in vitro model. We therefore study periodontal ligament (PDL) cells isolated from mice upper molars and compare them with murine tooth root cementum cells (OCCM) in a model of mechanical cell stress in order to simulate periodontal remodeling.
  • In part (II) we investigate physiological relevance for dental tissue remodeling in an in vivo model with TLR4-knockout mice via μPET/CT imaging using tracer for apoptosis, bone metabolism and inflammation, including their time dependent regulation in early and late phase of remodeling.
  • In part (III), we intend to define the correlation between findings from in vitro cell signaling, murine periodontal stimulation and mediator expression in human crevicular fluid samples. In line with our findings, we intend to map a tissue specific TLR4 signaling cascade for periodontium and cementum to define potential therapeutic targets for periodontal remodeling and disease.

OC1-3: Impact of stem cells on wound healing and integration of tissue engineered alveolar bone

Prof. Dr. rer. nat. Sabine Neuss-Stein 
Helmholtz Institute for Biomedical Engineering, Biointerface Group and Institute of Pathology

Univ.-Prof. Dr. rer. nat. Wilhelm Jahnen-Dechent         
Helmholtz Institute for Biomedical Engineering, Biointerface Group

 

Clinical evidence indicates that wounds in alveolar bone heal faster and more efficiently in the maxilla (upper jaw) than in the mandible (lower jaw). Alveolar bone differs in composition, with 23 percent  bone marrow and 46 percent lamellar bone in the upper jaw, and 16 percent bone marrow and 63 percent lamellar bone in the lower jaw. Differences concerning the endogenous stem cells (dental pulp stem cells, gingiva stem cells, mesenchymal stem cells and periodontal ligament cells), and their recruitment towards wounded areas and their differentiation potential may also regulate regeneration.

Human mesenchymal stem cells (MSC) are beneficial for wound healing and tissue regeneration of mesodermal tissue. MSC differentiate into the cementoblastic and osteoblastic lines, and exert their trophic function by secreting a variety of cytokines and growth factors. They are well-characterized and frequently used cells in bone tissue engineering approaches.

We propose to study periodontal ligament (PDL) cells isolated from third molars of the upper (u-PDL cells) and lower jaw (l-PDL cells) and compare them with MSC. We will analyze (i) their stem cell characteristics (ii), their trophic phenotype (via transcriptome and proteome analyses), (iii) their migration capacity and (iv) their cell fate within three-dimensional, hydrogel-based bone constructs. To trace PDL cell fate, we will generate stable, fluorescently labeled cell lines from the primary cells. Finally, we will improve established 3D models by using hydrogel-based, ECM containing co-culture systems using PDL cells and endothelial cells to build in vitro constructs with osteoblasts and capillary-like structures for future alveolar bone tissue engineering approaches.

OC1-4: Influence of the PCTAIRE Kinase CDK16 on osteoclast differentiation and biology in alveolar bone remodeling

Univ.-Prof . Dr. rer.. nat Bernhard Lüscher       
Institute of Biochemistry and Molecular Biology           

Dr. rer. nat. Antonio Sechi        
Institute of Biomedical Engineering / Department of Cell Biology

Orthodontic movement of teeth and periodontal diseases are associated with alveolar bone remodeling, which is executed by bone rebuilding osteoblasts and bone resorbing osteoclasts. For the latter osteoclasts attach onto the bone surface and create an isolated resorptive microenvironment. This requires the reorganization of the actin cytoskeleton to assemble a sealing zone around the resorptive microenvironment composed of podosomes. Moreover, membrane trafficking has to be reorganized to form bone-facing actin-based ruffled borders enriched in lysosomal membrane proteins. The coordination of these processes is still elusive.

CDK16 is a cytoplasmic cyclin-dependent kinase that is postulated to affect cytoskeletal processes. CDK16 is involved in the terminal steps of spermatogenesis to generate fertile sperms. In the brain CDK16 is suggested to regulate neurite outgrowth and in muscle CDK16 is involved in terminal differentiation. All these differentiation processes are accompanied by a massive reconstruction of the cellular content, shape and architecture, which requires reorganization of the actin cytoskeleton and membrane transport. To understand CDK16 at the molecular level, we performed a substrate screen and found that one quarter of the 41 identified potential CDK16 substrates have been described to regulate the actin cytoskeleton. In this proposal, we will focus on the regulation of actin cytoskeleton remodeling by the CDK16 substrates. We will investigate their phosphorylation-dependent regulation by CDK16 during osteoblast differentiation and bone resorption. To verify our findings in vivo we will use CDK16 and available substrates knock out mice to investigate their function for alveolar bone remodeling during orthodontic tooth movement.

This project provides a deeper understanding of the molecular mechanisms by which osteoclasts resorb bone and may lead to the development of new antiresorptive drugs targeting CDK16 or their substrates.

OC1-5: ALARMin MDS and periodontal diseas

Prof. Dr. Dr. Rebekka Schneider-Kramann 
Institute for Biomedical Engineerung, Department of Cell Biology

Dr. med. Martina Crysandt   
Department of Medicine IV

 

Descriptive studies demonstrated a link between periodontal inflammatory conditions and cancer. However, the underlying mechanisms remain unclear and haven’t been investigated using functional models. In this grant application, we will shed light on the functional association between periodontal inflammation and pre-leukemic conditions (MDS).

Preliminary evidence from our collaborative work with Michael Wolf supports a link between mutations in hematopoietic cells (pre-leukemia) and alterations in the oral cavity. MDS mutations led to degrading periodontal remodeling processes with an increase of inflammatory cells in the periodontal tissue.

Based on these data we hypothesize that the inflammation caused by MDS mutations in hematopoietic cells fuels the local inflammation in the periodontium. We propose that this leads to increased local but also systemic inflammation and a vicious cycle of periodontal and systemic inflammation and potentially clonal selection of mutant (pre-leukemic) hematopoietic cells which are potentially more resistant to inflammation than wild-type cells.

In aim 1, we thus plan to analyze the effect of chronic inflammation caused by periodontal inflammatory conditions on clonal selection and malignant transformation.

In aim 2, we will determine the influence of mutated hematopoietic cells on periodontal inflammatory bone degeneration.

Finally, in aim 3, we will determine if “inflammatory mediators” (in saliva/crevicular fluid can be used as a biomarker to detect pre-leukemic conditions.

OC1-6: Porphyromonas gingivalis – from genomics to inference of virulence signals on both local and systemic level in periodontal bone diseases

Dr. rer. nat. Mohamed Abdelbary        
Division of Oral Microbiology and Immunology

Univ.-Prof. Dr. rer. nat. Georg Conrads 
Division of Oral Microbiology and Immunology

Periodontitis is a biofilm-induced chronic inflammatory disease, which affects the tooth-supporting tissues including the alveolar bone. This continuing inflammation exerts a major impact on systemic health, as it increases e.g. a patient’s risk for atherosclerosis or diabetes. It is primarily the host inflammatory response that inflicts the irreversible damage to the periodontal tissues, but it is triggered by the sulcus-associated biofilm “red complex” bacterial species: Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis. The latter species transforms the normally symbiotic microbiota into a dysbiotic state. P. gingivalis has evolved sophisticated strategies to evade or subvert the host immune system.

We will isolate 20 strains of P. gingivalis from high-stage, high grade cases of periodontitis and compare these strains with 20 isolates from low-stage, low-grade cases. This comparison will be done on whole-genome level. We will determine e.g. the role of mobile genetic elements and core-gene mutations in the adaption of P. gingivalis to the inflamed subgingival sulcus or periodontal pocket leading to a vicious circle. Information on species interaction in the corresponding microbiota will be achieved by metagenome analysis.

Together with genome-information from publicly available databases we will generate the world’s largest genome database of this key-pathogen embedded in corresponding clinical and metagenome information. The main expected result is the elucidation of processes involved in P. gingivalis evolution to enhance our understanding of bacterial mechanisms and signaling underlying the development of virulence, both local and systemic.

 

OC1-7: Osteoclast activation by soluble mediators derived from inflamed mucosa

Univ.-Prof. Dr. rer. nat. Andreas Ludwig
Institute for Pharmacology and Toxicology       

Dr. rer. nat. Aaron Babendreyer           
Institute for Pharmacology and Toxicology

Infection or damage of oral mucosa triggers release of various mediators from epithelial cells. Soluble mediators become released either upon induction of biosynthesis, secretion of stored vesicular content or proteolytic shedding of surface expressed molecules. In this project we want to investigate the crosstalk between the soft mucosa and the hard jawbone. For this purpose, the nature and release mechanism of epithelial cell derived soluble mediators relevant for the modulation of osteoclast activity in the jawbone will be identified and investigated.

We will study conditioned media of epithelial cell lines and primary oral epithelial cells undergoing inflammatory or mechanic stress due to treatment with bacterial toxins, stretching or wounding. Release mechanisms for soluble mediators will be differentiated by inhibition of gene transcription, protein secretion or protease activity. We will study the potential of the epithelial supernatants to induce activation of cultured osteoclast cell lines and primary osteoclasts. Selected molecules will be investigated in detail, in terms of their induction, release mechanism, their receptor interaction and their pathway of osteoclast activation. This includes the transmembrane chemokine CX3CL1 that is induced in epithelial cells and shed by the proteolytic activity of ADAM family metalloproteinases. CX3CL1 mediates recruitment of monocytes and macrophages via its receptor CX3CR1 on osteoclasts.

We hypothesize that this pathway is relevant for osteoclast activation, which will be further studied in tissues of periodontitis patients and a murine model of ligation-induced gingivitis and periodontitis using knockout mice for ADAM proteases or CX3CR1.

OC1-8: Bone regeneration of typical morphological bone defect in periodontitis by mesenchymal stem cells of the pulp based on the regulatory influence of trauma related release of extracellular

PD Dr. med. Dr. med. dent. Ali Modabber     
Department of Oral and Maxillofacial Surgery  

Univ.-Prof. Dr. med. Frank Hildebrand  
Department of Trauma and Reconstructive Surgery

Mesenchymal stem cells (MSCs) have a positive effect on bone regeneration in experimental bone defect models. Therefore, dental pulp stem cells (DPSCs) and their extracellular vesicles (DPSC-EVs) are being considered as suitable therapeutic options in cases of impaired bone healing. These disturbances of bone regeneration are especially found in the context of relevant inflammatory reactions, as typically found in periodontitis and after severe trauma. This can result in bone loss or impaired fracture healing, both of which have a significant negative impact on the clinical outcome of the affected patients and represent a major therapeutic challenge for the treating physician. Therefore, the potential benefit of the therapeutic use of the DPSCs and DPSC-EVs under the above-mentioned conditions will be investigated in this study.

The aim is to analyze the influence of DPSCs and their EVs on the regeneration of bone defects and fracture healing. Furthermore, a potential inflammation modulating effect of DPSCs and EVs under inflammatory conditions in periodontal disease, isolated femur fractures, and polytrauma will be investigated. For this purpose, different scaffolds coated with DPSCs or DPSC-EVs will be used. The analyses include ELISAs, histological staining, μ CT scans and biomechanical tests.

OC1-9: Liver transplantation and its effects on the oral microbiome and periodontal health in combination with different dental prosthetic restoration

Univ.-Prof. Dr. med. dent. Stefan Wolfart       
Department of Prosthodontics and Biomaterials, Center of Implantology

Dr. med. Dr. rer. nat. Kai Markus Schneider    
Clinic for Gastroenterology, Metabolic Diseases and Internal Intensive Care Medicine

Emerging evidence suggests that there is a possible link between a dysbiotic oral microbiota and liver disease. While it is already well established that a dysbiotic oral microenvironment promotes oral diseases such as dental caries or periodontitis, recent data also highlight unfavorable oral microbiota composition and translocation of certain oral bacteria as drivers of liver disease progression.

Still, data on the pathophysiological crosstalk between oral and liver diseases and its interrelation with the oral and gut microbiota remain scarce. Liver transplant patients offer the unique opportunity to study how restoration of normal liver function affects oral microbiota, mucosal immunologic homeostasis and periodontal health. These patients provide valuable information on the impact that replacement of a diseased liver with a healthy liver has on the oral microbiota, gut microbiota and periodontal health in combination with different prosthetic restorations.

Based on a translational approach including a clinical human study with liver transplant patients as well as mechanistic preclinical studies in mouse models, the proposed project is designed to unravel clinically relevant pathophysiological connections between liver disease, oral microbiota and periodontal homeostasis.

The premise of this proposal is that a better understanding of the molecular circuits linking oral microenvironment with liver disease will guide efforts to identify future diagnostic and therapeutic targets.

OC1-10: ASC-based in vitro and in vivo bone tissue engineering for bone defect reconstruction

Dr. med. Rebekka Götzl
Department of Plastic Surgery, Hand Surgery, Burn Center       

Univ.-Prof. Dr. med. Justus P. Beier      
Department of Plastic Surgery, Hand Surgery, Burn Center

In an aging society, periodontal soft and hard tissue crosstalk, addressed by regenerative medicine and tissue replacement approaches, so called Tissue Engineering, plays an increasingly important role in periodontal disease. The incidence of a loss of alveolar bone mass and periodontal/ peri-implant degeneration will increase in an aging population. The use of human adipose derived stem cells (hASC) in Tissue Engineering is a promising approach in particular for alveolar bone tissue engineering for alveolar bone defect reconstruction. hASC of subcutaneous adipose tissue can be easily harvested in large quantities via liposuction, e.g. Human ASC are capable to differentiate into various cell types, including osteoblasts. In this study, we aim to attain a better understanding of the behavior of ageing primary hASC in alveolar bone tissue engineering.

In particular, we will analyze the donor´s age influence on viability, proliferation and osteogenic differentiation capacity of hACS and investigate the influence of different biophysical impacts on ageing ASC in vitro. Therefore, we will assess the impact of centrifugal gravity, hyperbaric therapy and hyperbaric oxygen (HBO) therapy as well as hypoxia. Furthermore, we will analyze the impact of pharmacological stimulation with different endocannabinoids on ageing hASC.

Subsequently, we will transfer the in vitro results in a subcutaneous in vivo, implanting dynamic pre-cultured hASC with an established osteogenic scaffold over 2 and 4 weeks in a small animal model as a first proof-of-concept study, in which we will analyze new bone formation and its vascularization.

OC1-11: Impact of chronic renal failure on periodontal bone disease and evaluation of potential treatment by recently identified and yet unknown calcification mediators

Univ.-Prof. Dr. rer. nat. Joachim Jankowski
Institute for Molecular Cardiovascular Research

Univ.-Prof. Dr. med. dent. Michael Wolf      
Department of Orthodontics and Craniofacial Orthopedics

The loss of kidney function leads to a reduction in kidney’s ability to filter metabolites, causing accumulation of uremic toxins in the blood. Uremia is a result of chronic kidney disease (CKD), and adverse outcomes include kidney failure, cardiovascular disease, and premature death. In addition, reduction of kidney function impairs regulation of acid-base balance, water and electrolyte balance, blood pressure and bone metabolism. Several reports demonstrated significant changes in soft and hard tissue homeostasis in the maxilla and mandibular facial system such as delayed tooth eruption, morphological alterations in the periodontium and alveolar bone, and deformation of tooth roots. Despite all evidences that CKD is closely associated with the onset and progression of periodontal degeneration, a detailed molecular understanding and potential therapeutically targets are missing.

The overall aim of this project is an assessment of morphological structure and chemical composition of changes in periodontal soft and hard tissue homeostasis in the maxilla and mandibular facial system in patients suffering from CKD and the effectiveness of remineralizing therapy. A major issue in this context are extra skeletal calcification processes, which indicate dysregulated mineral metabolism. We  are analyzing periodontal ligament cells and cementoblasts in their ability to mineralize in presence and absence of already known and yet unknown mediators, isolated from hemofiltrate of CKD patients. Furthermore, we  are analyzing their effect on the structure of alveolar bone and periodontal ligament in a mouse model and thus determine a potential role on periodontal degeneration.

OC1-12: Evaluation of endodontic/periodontal interface and impact on alveolar bone

Univ.-Prof. Dr. med. dent. Andreas Braun 
Clinic for Operative Dentistry, Periodontology and Preventive Dentistry    

Dr. med. dent. Johannes-Simon Wenzler         
Clinic for Operative Dentistry, Periodontology and Preventive Dentistry

The development of new medical therapies increasingly involves the use of biocompatible materials to replace destroyed or lost natural tissues. In addition to a possible defense reaction to the material used, the unavoidable interface areas between biological tissue and artificial material has a major impact on possible healing disorders. Especially an insufficient endodontic/periodontal interface can cause a penetration of bacteria, remnants of necrotic tissue or inflammatory mediators remaining in the dentinal tubules in periapical tissues and thus causing alveolar bone inflammation. Therefore, the objective of the project is the development of a universal analysis system for testing interfaces for leaks, diffusion and emanation, suitable to assess the endodontic/periodontal interface.

The test system uses a high vacuum to quantitatively assess even smallest particles and analyze them by mass spectrometry. In the diffusion test, a measuring chamber filled with test gas shall be sealed by a test specimen, consisting of a hybrid structure of root dentin and endodontic sealing material. The measuring arrangement is then connected to the high-vacuum chamber and evacuated. The test gas diffusing through the test specimen is then quantitatively detected by a mass spectrometer. During the emanation test, the gases and evaporable chemical compounds emitted from the material sample can be fed to a mass spectrometer and determined both qualitatively and quantitatively.

The study intends to generate a standardized simulation and test set-up for endodontic/periodontal interfaces to assess and optimize hybrid structures of root dentin and endodontic sealing material to avoid affecting surrounding alveolar bone tissue.

OC1-13: In vitro and in silico investigation of mechano-regeneration of subchondral bone using an artificial neuronal network for cartilage - alveolar bone vs. long bone

Dr.-Ing. Jörg Eschweiler
Department of Orthopaedic Surgery

Univ.-Prof. Dr. med. Markus Tingart     
Department of Orthopaedic Surgery

The aim of this approach is to answer if there can be any optimization of substrate stiffness and mechanical loading in order to induce the mechano-regeneration of the subchondral bone of the alveolar bone in comparison to the long bone for cartilage.

We improve an in-house bioreactor system with parallel cultivation chambers which enables to perform dynamic experiments with different parameters. Based on functionalized membranes with a diversity in material stiffness, cells will be seeded on them. Human alveolar bone (HAB), human long bone (HLB), and human periodontal ligament (HPL) cells will be cultured on these artificial silicone membranes which possess different stiffness and will be exposed to a different combination of mechanical loading. After mechanical stimulation, differentiation markers will be analyzed by real-time polymerase chain reaction (PCR) in order to control the osteogenic behavior and differentiation, respectively, of the cells. Cell density and arrangement of cells will be determined using histological methods. Withal, substrate stiffness of scaffolds will be measured prior to dynamic experiments as well as after dynamic experiments in order to determine the alteration in stiffness. Finally, the data gathered from experiments will be used to train an artificial neuronal network (ANN) which can be used to predict and improve the mechanical properties of bone tissue. Sensitiveness analysis method in ANN will be used to determine the significance of different input parameters on the mechanical properties of the scaffold. The major question here is, if it will be possible to create an ANN model which includes cellular process, in our case, is osteogenesis, as an input values.

OC1-14: Unraveling the effect of dynamic forces on regeneration capacity of periodontal ligament stem cells embedded in 3D bone‐like ECM

Univ.-Prof. Dr. Ing. Horst Fischer          
Zahnärztliche Werkstoffkunde und Biomaterialforschung (ZWBF)         

Dr. rer. nat. Sanja Aveic
Zahnärztliche Werkstoffkunde und Biomaterialforschung (ZWBF)

Periodontal tissue is a complex structure composed of alveolar bone, gingiva, cementum, and per-iodontal ligament (PDL). Multipotent periodontal ligament stem cells (PDLSC) can be isolated from the PDL and have been exploited for differentiation towards the alveolar bone. Their proper func-tion is important for the periodontal homeostasis maintenance during orthodontic tooth movement (OTM). During this movement, mechanical stresses are applied to the teeth and diffused to the al-veolar bone through the PDL. Mechanical forces, including compression and stretching, continu-ously occur during jaw movement and occlusal forces. However, a study of the molecular back-ground during dynamic interaction between multicellular components of the periodontal tissue is hardly achievable due to a lack of adequate in vitro models able to mimic physiological events on-going during OTM.

Therefore, we propose a novel tissue-engineered approach to explore the effect of dynamic load-ing on PDLSC. For that purpose, we will apply three-dimensional (3D) hydrogel structures laden with PDLSC, fibroblasts, and endothelial cells. By applying stretching mechanism forces we will simulate conditions found in vivo. This approach will allow us to examine the effects of mechanical forces on cell proliferation, differentiation, and interaction between the cells and their extracellular matrix. The expected findings will contribute to a better understanding of how physiological forces influence PDLSC, deriving osteoblast and fibroblast cells and will improve our knowledge of the importance of endothelial cells for the intercellular crosstalk inside the alveolar bone.